WO2011017107A2 - Administration virale aidée par microbulle - Google Patents

Administration virale aidée par microbulle Download PDF

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Publication number
WO2011017107A2
WO2011017107A2 PCT/US2010/043403 US2010043403W WO2011017107A2 WO 2011017107 A2 WO2011017107 A2 WO 2011017107A2 US 2010043403 W US2010043403 W US 2010043403W WO 2011017107 A2 WO2011017107 A2 WO 2011017107A2
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tumor
mda
virus
cancer
cells
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PCT/US2010/043403
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WO2011017107A3 (fr
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Paul B. Fischer
Devanand Sarkar
Rupesh Dash
Belal Mohammed Azab
Xiang-Yang Wang
Pier Paolo Claudio
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Virginia Commonwealth University
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Publication of WO2011017107A2 publication Critical patent/WO2011017107A2/fr
Publication of WO2011017107A3 publication Critical patent/WO2011017107A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10371—Demonstrated in vivo effect
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • Prostate cancer is the most common cancer and the second leading cause of cancer related deaths in men in the United States (Damber, JE, and Aus, G (2008). Prostate cancer. Lancet 371 : 1710-1721). At present, no effective therapy is available for metastatic prostate cancer (PC) (Moon, C, Park, JC, Chae, YI, Yun, JH, and Kim, S (2008). Current status of experimental therapeutics for prostate cancer. Cancer Lett 266: 116-134). Advanced PC is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or BcI-XL (Shi, XB, Gumerlock, PH, and deVere White, RW (1996).
  • PC metastatic prostate cancer
  • Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24.
  • the Melanoma differentiation associated gene7/ interleukin-24 (mda-7/IL-24), is a secreted cytokine having broad-spectrum, cancer-selective, apoptosis- inducing properties that profoundly inhibits prostate cancer cell growth (Lebedeva, IV, et al. (2003). Melanoma differentiation associated gene-7, mda 7/interleukin-24, induces apoptosis in prostate cancer cells by promoting mitochondrial dysfunction and inducing reactive oxygen species. Cancer Res 63: 8138-8144).
  • Adenovirus (Ad)-mediated delivery o ⁇ mda- 7/IL-24 (Ad.mda-7) has shown dramatic anti-tumor effects in animal models and in clinical trials (Fisher, PB (2005) Is mda-7/IL-24 a "magic bullet" for cancer? Cancer Res 65: 10128- 10138; Fisher, PB, et al. (2003) mda-7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic. Cancer Biol Ther 2: S23-37; Lebedeva, IV, et al. (2007).
  • mda-7/IL-24 novel anticancer cytokine: focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience (Review). IntJ OncoBl: 985-1007; Lebedeva, IV, et al. (2005) mda-7/IL-24: exploiting cancer's Achilles' heel. MoI Ther 11 : 4-18). However, forced overexpression of Bcl-2 or BcI-XL renders prostate cancer cells resistant to Ad.mda-7 (Lebedeva, IV, et al. (2003).
  • Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24. Oncogene 22: 8758-8773).
  • CRCA conditionally replication-competent adenovirus
  • mda-7 VIL-24 Ad.PEG-El A-mda-7-7
  • BcIXL BcIXL
  • the invention provides methods of targeting a virus to cancer cells in an
  • the method comprises administering a selectively replicating virus to the immunocompetent animal, wherein the virus is
  • a suspension of microbubbles wherein the surface of the suspension does not include any virus; and disrupting the microbubbles administered to the animal in a location of the animal comprising cancer cells, wherein the virus selectively replicates in the cancer cell.
  • the animal is a human.
  • the cancer cell is killed as a result of replication or gene expression of the virus in the cancer cell.
  • the virus further expresses a chaperone protein, wherein the chaperone protein presents killed cancer cell antigens to the animal immune system.
  • the chaperone protein is Grpl70.
  • the virus comprises a cancer-specific promoter operably linked to at least one viral gene necessary for viral replication, thereby rendering the virus selectively replicating.
  • the promoter is a human PEG-3 promoter.
  • the virus further expresses a heterologous polynucleotide, wherein the heterologous polynucleotide encodes mda-7.
  • expression of the heterologous polynucleotide is under the control of a human PEG-3 promoter.
  • the virus is an adenovirus.
  • the virus and/or suspension of microbubbles have altered tropism.
  • the invention also provides for suspensions of microbubbles, the suspension including a selectively replicating virus wherein the virus is encompassed in a suspension of microbubbles and the surface of the suspension does not include any virus.
  • the virus selectively replicates in cancer cells.
  • a cancer cell is killed as a result of replication or gene expression of the virus in the cancer cell, wherein the virus further expresses a chaperone protein, wherein the chaperone protein is capable of presenting killed cancer cell antigens to the animal immune system.
  • the chaperone protein is Grpl70.
  • the virus comprises a cancer-specific promoter operably linked to at least one viral gene necessary of viral replication, thereby rendering the virus selectively replicating.
  • the promoter is a human PEG-3 promoter.
  • the virus further expressed a heterologous polynucleotide, wherein the heterologous polynucleotide encodes mda-7.
  • expression of the heterologous polynucleotide is under the control of a human PEG-3 promoter.
  • the virus is an adenovirus.
  • the virus and/or suspension of microbubbles have altered tropism.
  • the invention also provides pharmaceutical compositions comprising the suspension as described above.
  • Figure 1 illustrates A) Schematic representation of the microbubble delivery of Ad- GFP complexes and US release in a tumor target site of the mouse.
  • Figure 2 illustrates ultrasound imaging and US contrast enhancement of in vivo transduced DU- 145 tumor xenografts.
  • Panel A B-Mode US imaging of a tumor before MB contrast agent injection.
  • Panel B B-Mode US imaging of the same tumor depicted in panel A following injection of microbubbles/ Ad-GFP complexes. MBs cavitation within the targeted tumor dramatically enhances the tumor image within the US field of view.
  • Figure 3 illustrates growth curves and Western blot analysis of large DU- 145 and DU-Bcl-xL tumor xenografts treated with microbubble encapsulated Ad-GFP, Ad.mda-7 or CTV (Ad.PEG-El A-mda-7) and treated with US in the right tumor.
  • Subcutaneous tumor xenografts from DU- 145 and DU-Bcl-xL were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/ Ad complexes during a course of 4 wks.
  • Tumor treatments were initiated when tumors reached a size of 250 - 350 mm . Arrows point at tumors and asterisks point at treatment times.
  • Figure 4 illustrates growth curves of DU-145 tumor xenografts treated with microbubble/ultrasound guided Ad-GFP, Ad.mda-7, or CTV.
  • Subcutaneous tumor xenografts from DU-145 cells were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/ Ad complexes during a course of 4 weeks. Tumor treatments were initiated when tumors reached a size of 25-35 mm .
  • FIG. 5 illustrates Colorimetric TUNEL assay of DU-145 tumor xenografts treated with microbubble/ultrasound guided Ad-GFP, Ad.mda-7, or CTV.
  • Subcutaneous tumor xenografts from DU-145 cells were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/Ad complexes during a course of 4 weeks. Tumors were removed, fixed, sectioned and stained to determine levels of double-stranded DNA breaks (TUNEL). Microscopy for TUNEL sections was under visible light at 4OX magnification (a)
  • A-B TUNEL staining of left and right side DU-145 tumors following systemic injection of Ad-GFP-microbubble plus US treatment of the tumor on the right side.
  • C-D TUNEL staining of left and right side DU-145 tumors following systemic injection of Ad.m ⁇ i ⁇ -7-microbubble plus US treatment of the tumor on the right side.
  • E-F TUNEL staining of left and right side DU-145 tumors following systemic injection of C7T-microbubble plus US treatment of the tumor on the right side..
  • Figure 6 illustrates growth curves of therapy resistant DU-Bcl-xL tumor xenografts treated with microbubble/ultrasound guided Ad-GFP, Ad.mda-7, or CTV.
  • Subcutaneous tumor xenografts from DU-Bcl-xL cells were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/Ad complexes during a course of 4 weeks. Arrows point at tumors and asterisks point at treatment times.
  • FIG. 7 illustrates colorimetric TUNEL assay of therapy resistant DU-Bcl-xL tumor xenografts treated with microbubble/US guided Ad-GFP, Ad.mda-7, or CTV.
  • Subcutaneous tumor xenografts from DU-Bcl-xL cells were established in athymic nude mice in both right and left flanks, and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/ Adenoviral complexes during a course of 4 weeks. Tumors were removed fixed, sectioned and stained to determine levels of double- stranded DNA breaks (TUNEL). Microscopy for TUNEL sections was under visible light at x40 magnification (a representative of 3 separate tumors). A-B) TUNEL staining of left and right side DU-Bcl-xL tumors following Ad-GFP-microbubble treatment.
  • C-D TUNEL staining of left and right side DU-Bcl-xL tumors following Ad.m ⁇ i ⁇ -7-microbubble treatment.
  • E-F TUNEL staining of left and right side DU-Bcl-xL tumors following C7T-microbubble treatment.
  • Figure 8 illustrates immunohistochemical analysis of DU- 145 and therapy resistant DU-Bcl-xL tumor xenografts treated with microbubble/US guided Ad-GFP, Ad.mda-7, or CTV.
  • Subcutaneous tumor xenografts from DU- 145 or DU-Bcl-xL cells were established in athymic nude mice in both right and left flanks, and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/Adenoviral complexes during a course of 4 weeks. Tumors were removed fixed, sectioned and immunostained to determine levels of ElA expression.
  • Figure 9 illustrates B-Mode ultrasound imaging of DU- 145 tumor xenografts treated with microbubble/ultrasound guided Ad.mda-7 and therapy resistant DU-Bcl-xL tumor xenografts treated with microbubble/ultrasound guided CTV.
  • Subcutaneous tumor xenografts from DU- 145 and DU-Bcl-xL cells were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated microbubble/ Adenoviral complexes during a course of 4 weeks.
  • Tumor volumes were determined by measuring twice a week the tumors with either a caliper or by ultrasound measurements of the tumor axes.
  • FIG. 10 illustrates growth curves of control DU-145 tumor xenografts injected i.v. using unprotected Ad-GFP, Ad.mda-7 or C7T (Ad.PEG-ElA-md ⁇ -7) and treated or not with US.
  • Subcutaneous tumor xenografts from DU-145 were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated Ads during a course of 4 wks. Tumor treatments were initiated when tumors reached a size of 150 - 200 mm 3 . Asterisks point at treatment times.
  • Ad.m ⁇ i ⁇ -7-injected, but not sonoporated DU-145 tumor volumes The data represent mean ⁇ s.d. with at least 5 mice in each group.
  • Figure 11 illustrates growth curves of control DU-Bcl-xL tumor xenografts injected i.v. using unprotected Ad-GFP, Ad.mda-7 or C7Y (Ad.PEG-ElA-m ⁇ i ⁇ -7) and treated or not with US.
  • Subcutaneous tumor xenografts from DU-Bcl-xL were established in athymic nude mice in both right and left flanks and only tumors on the right side were sonoporated following tail vein injection of the indicated Ads during a course of 4 wks. Tumor treatments were initiated when tumors reached a size of 150 - 200 mm 3 . Asterisks point at treatment times.
  • FIG. 12 illustrates adenovirus-mediated expression of secretable grpl70 in TRAMP-C2 tumor cell.
  • A Schematic representation of adenovirus vector encoding a secretable form of grpl70 (Ad.sgrpl70).
  • the COOH-terminal KNDEL signal was deleted from mouse grpl70 cDNA to produce the secreted form of grpl70.
  • the His-tagged sgrpl70 gene under the control of a constitutively active cytomegalovirus promoter/enhancer (CMV) was inserted into the replication incompetent adenoviral vector, in which the E1/E3 sequences have been deleted. Inverted terminal repeats (ITR), which flank the E1/E3 deleted genome, are necessary for the replication of adenoviral DNA.
  • CMV constitutively active cytomegalovirus promoter/enhancer
  • ITR Inverted terminal repeats
  • the TRAMP-C2 cells were infected with or without Ad.sgrpl70 at different MOIs. Supernatants were collected from the infected cells at different time points and analyzed for the expression of sgrpl70 using antibodies against grpl70 (B) or His-tag (C).
  • FIG. 13 illustrates adenovirus-mediated mda-1 inhibits TRAMP-C2 tumor cell growth by inducing apoptosis.
  • A. Ad.mda-7 infection suppresses proliferation of C2 tumor cell in vitro. C2 cells were infected with Ad.sgrpl70, Ad.mda-7 at different MOIs or left untreated. Protein lysates (50 ⁇ g) were run on 12% SDS-PAGE and stained with anti-mda- 7/IL-24 monoclonal antibody (1 :2,000) (top). C2 cells were infected with Ad.GFP, Ad.mda- 7 at a MOI of 300 or left untreated. Cell proliferation was analyzed using MTT assay (bottom). B.
  • Ad.mda-7 infection induces C2 tumor cell apoptosis.
  • TRAMP-C2 tumor cells were infected with Ad.GFP, Ad.mda-7 or left untreated. Cells were collected at 72 h after treatment and stained with FITC-labeled Annexin-V. The percentage of Annexin-V positive cells was analyzed by flow cytometry (* /? ⁇ 0.01, Ad.mda-7 versus Ad.GFP or untreated control).
  • C. Ad.mda-7 infection promotes PARP cleavage in C2 tumor cells.
  • C2 cells were infected with or without Ad.GFP or Ad.mda-7.
  • Ad.mda-7 induces apoptosis in tumor cell but not in normal cells.
  • TRAMP-C2 prostate tumor cells or DC 1.2 dendritic cells were infected with Ad.mda-7.
  • Cells were stained with FITC-labeled Annexin-V 48 h later and examined using FACS (dot line - control; solid line - Ad.mda-7 treated cells).
  • FIG 14 illustrates intratumoral administration of adenovirus encoding mda-7/lL- 24 and secretable grpl70 induces a systemic antitumor response.
  • Ad.mda-7 or Ad.sgrpl70 D.
  • C2 tumor cells were inoculated s.c. into the right flank of mice. When tumor volume reached the size of 5 mm in diameters, mice were treated with Ad.GFP, Ad.mda-7, Ad.sgrpl70 or Ad.mda-7 plus Ad.sgrpl70. Tumors were removed by surgery one week after the last treatment.
  • FIG. 15 illustrates intratumoral delivery o ⁇ Ad.mda-7 and Ad.sgrpl70 promotes antigen-specific immune response.
  • C2 tumor cell line stably expressing OVA (C2-OVA). TRAMP-C2 cells were transduced with pcDNA-OVA using FuGENE trans fection reagent and selected in G418 (1 mg/ml)-containing medium.
  • OVA OVA expression was analyzed using RT-PCR assays. Primers of GAPDH were used as an internal control.
  • Splenocytes from the treated mice were harvested one week or three weeks after the last injection.
  • Cells were re-stimulated with OVA 2 57-264 (SIINFEKL) in vitro for 5 d in the presence of IL-2 (10U/ml).
  • the CD8 + T- cells at different E:T ratios in triplicate were analyzed for cytotoxic activity using 51 Cr- labeled C2-OVA as targets. Data are representative of three experiments.
  • FIG. 16 illustrates CD8 + T-cells contribute to the antitumor activities mediated by the combined therapies in vivo.
  • A. Depletion of CD8 + T-cell subset abolishes antitumor immunity. Male C57BL/6 mice (n 6) with established C2-OVA tumors were depleted of CD4 + , CD8 + T-cells by i.p. injection of GKl.5, 2.43 mAb respectively. Isotype-matched antibodies were used as controls. (CD8 depletion versus IgG control, p ⁇ 0.01).
  • B. The combined in situ therapies results in a tumor-specific immune response.
  • FIG. 17 illustrates antitumor immunity remains intact following separate administration of Ad.mda-7 and Ad.sgrpl70.
  • Mice with established C2 tumors were treated with Ad.mda-7, Ad.sgrpl70 together with Ad.mda-7 (T).
  • mice One group of mice was treated with Ad.mda-7 and Ad.sgrpl70 separately on different days (S).
  • C Both therapeutic regimens elicit similar levels of antigen-specific T-cells. Splenocytes isolated from mice one week after the last treatment and stimulated with OVA257-264- IFN- ⁇ production was measured using an ELISPOT assay (* and ** p ⁇ 0.01, versus control).
  • D T-effector cells from mice treated with Ad.mda-7 and Ad.sgrpl70 together displayed an increased cytolytic activity compared with cells from mice treated with the two therapeutic agents separately.
  • suspensions systemically to an immunocompetent animal in microbubble suspensions.
  • the suspensions are treated such that the suspensions lack virus on the surface of the suspensions.
  • suspension of microbubbles in which virus was not removed from the surface of the suspension did not result in significant systemic delivery.
  • inclusion of virus on the surface of the microbubble suspension raises an immune system response in immunocompetent animals that prevents successful delivery of the virus to a desired location in the animal.
  • the inventors have found that removal of virus from the surface of the suspension (e.g., by an initial incubation with complement, thereby removing the virus on the surface of the microbubbles) results in significantly improves systemic delivery. Once systemic delivery of the microbubbles is achieved, the microbubbles can be disrupted locally (e.g., with ultrasound), thereby releasing virus at only a desired location.
  • the microbubbles can be disrupted locally (e.g., with ultrasound), thereby releasing virus at only a desired location.
  • PEG-3 refers to Progression Elevated Gene-3.
  • Mda-7 is Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/ ⁇ L- 24). Mda-7 is a novel member of the IL-10-related cytokine gene family. It is a cancer- specific, apoptosis-inducing gene with broad-spectrum antitumor activity.
  • Grpl70 is 170 kDa glucose-regulated protein.
  • Grpl70 is an endoplasmic reticulum resident protein that shares some sequence homology with both the hsp70 and hspl 10 heat shock protein (hsp) families, yet is representative of a third and unique family of stress proteins.
  • the term "immunocompetent” as used herein refers to the ability to produce a normal immune response, i.e., antibody production and/or cell-mediated immunity, following exposure to an antigen. In an immunocompetent system, there are mature B cells and T cells capable of recognizing and responding to an antigen. An immunocompetent animal has a functional thymus.
  • nude mice are not immunocompetent.
  • Immunocompetent systems are capable of immunoglobulin and T-cell receptor gene recombination.
  • animals and human having mutated or knockout RAGl and/or RAG2 genes are not immunocompetent because immunoglobulin and T-cell receptor gene recombination are compromised.
  • the methods of the invention include administering a virus (including but not limited to a selectively replicating virus) that is encompassed in a suspension of
  • microbubbles carry the virus to a site in the subject where the virus is released from the microbubble.
  • microbubbles are about 0.1 to lO.mu. in diameter and in a fluid medium.
  • the microbubble diameter is about 2.5 to 4 ⁇ m. This is small enough to prevent entrapment within the pulmonary capillary bed (ranging from 5 to 8 ⁇ m in diameter), but big enough to entrap and protect viral vectors, e.g., adenovirus, from the environment.
  • the microbubbles contain a blood- insoluble gas. Generally, any blood-insoluble gas which is nontoxic and gaseous at body temperature can be used.
  • the insoluble gas should have a diffusion coefficient and blood solubility lower than nitrogen or oxygen, which diffuse in the internal atmosphere of the blood vessel.
  • useful gases are the noble gases, e.g. helium or argon, as well as fluorocarbon gases and sulfur hexafluoride.
  • perfluorocarbon gases such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and perfluoropentane, are used. It is believed that the cell membrane fluidizing feature of the perfluorobutane gas enhances cell entry for drugs on the surface of bubbles that come into contact with denuded vessel surfaces, as described further below.
  • the microbubbles contain high-molecular weight gasses with less solubility and diffusivity, which improves microbubble persistence and allows passage through the microcirculation.
  • microbubbles are injected in peripheral veins, and the microbubbles can re-circulate through the systemic circulation numerous times, surviving for several minutes within the bloodstream.
  • the microbubbles protect the viruses from rapid degradation by the immune system, thus allowing for intravenous injection rather than direct target organ delivery by catheter-based approaches or operative bed injection. This is beneficial for cancer gene therapy of potentially inaccessible tumors, because the microbubbles may also limit the amount of inflammatory response to the viruses and may allow repeated injections.
  • Targeting ligands on the surface of microbubbles permit the selective accumulation of these particles in the areas of interest, such as up-regulated levels of receptor/prognostic marker molecules on vascular endothelium or tumor cells.
  • the gaseous microbubbles can be stabilized by a fluid filmogenic coating, to prevent coalescence and to provide an interface for binding of molecules to the microbubbles.
  • the fluid is an aqueous solution or suspension of one or more components selected from proteins, surfactants, and polysaccharides.
  • the components are selected from proteins, surfactant compounds, and polysaccharides.
  • Suitable proteins include, for example, albumin, gamma globulin, apotransferrin,
  • the component is a human protein, e.g. human serum albumin (HSA).
  • HSA human serum albumin
  • a mixture of HSA and dextrose is used.
  • Conventional surfactants include compounds such as alkyl polyether alcohols, alkylphenol polyether alcohols, and alcohol ethoxylates, having higher alkyl (e.g. 6-20 carbon atom) groups, fatty acid alkanolamides or alkylene oxide adducts thereof, and fatty acid glycerol monoesters.
  • Surfactants particularly intended for use in microbubble contrast agent compositions are disclosed, for example, in Nycomed Imaging patents U.S. Pat. No.
  • filmogenic synthetic polymers may also be used; see, for example, U.S. Pat. No. 6,068,857 (Weitschies) and No. 6,143,276 (Unger), which describe microbubbles having a biodegradable polymer shell, where the polymer is selected from e.g. polylactic acid, an acrylate polymer, polyacrylamide, polycyanoacrylate, a polyester, polyether, polyamide, polysiloxane, polycarbonate, or polyphosphazene, and various combinations of copolymers thereof, such as a lactic acid-glycolic acid copolymer.
  • the polymer is selected from e.g. polylactic acid, an acrylate polymer, polyacrylamide, polycyanoacrylate, a polyester, polyether, polyamide, polysiloxane, polycarbonate, or polyphosphazene, and various combinations of copolymers thereof, such as a lactic acid-glycolic acid copolymer.
  • compositions have been used as contrast agents for diagnostic ultrasound, and have also been described for therapeutic applications, such as enhancement of drug penetration (Tachibana et al, U.S. Pat. No. 5,315,998), as thrombolytics (Porter, U.S. Pat. No. 5,648,098), and for drug delivery.
  • the latter reports require some external method of releasing the drug at the site of delivery, typically by raising the temperature to induce a phase change (Unger, U.S. Pat. No. 6,143,276) or by exposing the microbubbles to ultrasound (Unger, U.S. Pat. No. 6,143,276; Klaveness et al, U.S. Pat. No. 6,261,537;
  • the microbubbles are a suspension of perfluorocarbon- containing dextrose/albumin microbubbles known as PESDA (perfluorocarbon-exposed sonicated dextrose/albumin).
  • PESDA perfluorocarbon-exposed sonicated dextrose/albumin
  • Human serum albumin (HSA) is easily metabolized within the body and has been widely used as a contrast agent.
  • the composition may be prepared as described in co-owned U.S. Pat. Nos. 5,849,727 and 6,117,858. Briefly, a dextrose/albumin solution is sonicated while being perfused with the perfluorocarbon gas.
  • the microbubbles are formed in an N2-depleted or N2-free, environment, typically by introducing an N2-depleted (in comparison to room air) or N2-free gas into the interface between the sonicating horn and the solution. Microbubbles formed in this way are found to be significantly smaller and stabler than those formed in the presence of room air. See e.g. Porter et al., U.S. Pat. No. 6,245,747.
  • the microbubbles encompass the virus, as described below.
  • the microbubble suspension is incubated, with agitation if necessary, with the virus.
  • the incubation can be carried out at room temperature, or at moderately higher temperatures, as long as the stability of the drug or the microbubbles is not compromised.
  • the viral suspension can be prepared using standard laboratory methods. Tissue culture cell lines or a suitable animal can be used to propagate the virus. Traditionally, cells have been used for viral propagation. The cells used have to be readily infected by the viruses. The cell lines then amplify the amount of virus, and in many cases die as a consequence of the viral infection producing characteristic cytopathic effects in the cell monolayer. After a virus is propagated in either cell culture or in a suitable animal, the infectivity titer of the virus material is obtained. The infectivity titer can be determined in vivo by inoculating increasing dilutions of the virus material to a susceptible host animal, such as laboratory mice.
  • the infectivity titer can calculated by suing either Reed-Muench or Karber formula.
  • the infectivity titer is the reciprocal of highest dilution showing 50% mortality in the inoculated mice and expressed as LD50 /ml.
  • adenovirus is produced in HEK293 cells and infection titers are determined by plaque tittering on 293 cells. The virus can then be concentrated and purified, e.g., AdenoPACK Maxi columns.
  • HEK293 cells are human embryonic kidney cells that contain and express the essential El region of the viral genome. This complementation, which is necessary because El is deleted in the vectors, does not occur in other cell types and is a safety feature for gene therapy purposes.
  • microbubbles hide the virus from a competent immune system, which would otherwise recognize and kill the virus. Therefore, treatment of the microbubble suspensions to remove virus on the surface of the suspension may result in significantly improved systemic delivery of the microbubbles.
  • the microbubble suspensions are treated such that the suspensions lack virus on the surface of the suspensions.
  • the microbubble suspension is incubated with complement, thereby removing viruses on the surface of the microbubble suspension.
  • Any virus that has been used for gene therapy is suitable for use in this invention.
  • viruses e.g., lentivirus, adenoviruses, adeno-associated viruses and herpes simplex viruses, can be used as described herein.
  • Adenoviruses are medium-sized (90-100 nm), nonenveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Because of their size, they are able to be transported through the endosome (i.e. envelope fusion is not necessary).
  • the virion also has a unique "spike" or fiber associated with each penton base of the capsid (see picture below) that aids in attachment to the host cell via the coxsackie- adeno virus receptor on the surface of the host cell.
  • the adenovirus genome is linear, non- segmented double stranded (ds) DNA which is between 26 and 45 Kbp.
  • the viral genome has a terminal 55 kDa protein associated with each of the 5' ends of the linear dsDNA, these are used as primers in viral replication and ensure that the ends of the virus' linear genome are adequately replicated.
  • Adenoviruses are able to replicate in the nucleus of mammalian cells using the host's replication machinery.
  • Entry of adenoviruses into the host cell involves two sets of interactions between the virus and the host cell. Entry into the host cell is initiated by the knob domain of the fiber protein binding to the cell receptor.
  • the two currently established receptors are: CD46 for the group B human adenovirus serotypes and the coxsackievirus adenovirus receptor (CAR) for all other serotypes. This is followed by a secondary interaction, where a specialized motif in the penton base protein interacts with an integrin molecule. It is the co-receptor ⁇ v integrin interaction that stimulates internalization of the adenovirus.
  • Binding to ⁇ v integrin results in endocytosis of the virus particle via clathrin-coated pits. Once the virus has successfully gained entry into the host cell, the endosome acidifies and releases the virion into the cytoplasm. The virus is then transported to the nuclear pore complex whereby the adenovirus particle disassembles. Viral DNA is subsequently released which can enter the host cell's nucleus. Thus viral gene expression can occur and new virus particles can be generated.
  • the virus is a modified adenovirus having a tissue or disease specific promoter (e.g., the PEG-3 promoter) operably linked to the ElA (and ElB) gene(s) which further comprises an additional active transcriptional unit expressing a heterologous (non-adenoviral) gene of interest.
  • the gene of interest may encode a secreted product or a non-secreted product.
  • modified viruses are referred to herein as "Terminator Viruses”.
  • the gene of interest is comprised in the E3 gene of adenovirus. Details of such viruses can be found in, e.g., US Patent Publication No. 2008/0213220.
  • a gene of interest may be, for example and not by way of limitation, a gene that augments immunity (in a subject to whom the virus is administered), such as IFN-. alpha., IFN-.beta., IFN-. gamma., IL-2, IL-4, IL-12 etc., a gene involved in innate immune system activation such as mda-5 (Kang et al, 2002 Proc Natl Acad Sci USA. 99(2):637-42), RIG-I (Heim, 2005, J Hepatol.
  • a gene that has an anti-cancer effect including genes with anti-proliferative activity, anti-metastatic activity, anti-angiogenic activity, or pro- apoptotic activity, such as mda-7/IL-24 (Sarkar et al. (2002) Biotechniques Suppl: 30-39; Fisher et al. (2003) Cancer Biol Ther 2:S23-37), TNF-alpha (Anderson et al. Curr Opin Pharmacol (2004) 4(4):314-320), IFN-beta (Yoshida et al, (2004) Cancer Sci 95(11):858- 865), p53 (Haupt et al. Cell Cycle (2004) 3(7):912-916), BAX (Chan et al. Clin Exp
  • sFGFR soluble fibroblast growth factor receptor
  • RNAi or antisense VEGF Qui et al. Hepatobiliary Pancreat Dis Int (2004) 3(4):552-557
  • antisense or RNAi mda-9/syntenin Sarkar et al.
  • a gene that renders an infected cell detectable such as green fluorescent protein (or another naturally occurring fluorescent protein or engineered variant thereof), .beta.-glucuronidase, .beta.-galactosidase, luciferase, and dihydrofolate reductase, or a gene which enhances radiotherapy including but not limited to p53 (Haupt et al. Cell Cycle (2004) 3(7):912-916), GADD34 (Leibermann et al. Leukemia (2002) 16(4):527-41), the sodium iodide symporter (for thyroid cancer) (Mitrofanova et al. Clin Cancer Res (2004)
  • a modified adenovirus having a PEG-3 promoter operably linked to the ElA and ElB genes and comprising an additional active transcriptional unit expressing a heterologous gene of interest may be utilized to deliver a therapeutic amount of an anti-inflammatory, anti-allergic or antiviral gene product either systemically or at a specific target site in a human subject or non-human animal.
  • Non- limiting examples of such genes include IFN- ⁇ or IFN- ⁇ (Markowitz, Expert Opin Emerg Drugs (2004) 9(2): 363 -374) to treat an inflammatory condition or for anti-viral therapy
  • a modified adenoviral vector may comprise, as a gene of interest, a gene having a product that enhances, in a subject having a cancer, the immune response of the subject to the cancer.
  • Suitable genes of interest include, but are not limited to, genes encoding tumor-associated antigens recognized by the immune system, such as gplOO, PSA, EGFR, CEA, HER-2/neu, CO17-la, MUC-I, gp72/CD55, gastrin, beta-HCG, alpha-fetoprotein, heat shock protein (gp96), etc. (Mocellin et al.
  • genes of interest encoding costimulatory ligands such as B7-H3 (Luo et al. (2004) J Immunol 173(9):5445-5450), GM-CSF/IL-2 fusion protein (Stagg et al. (2004) Cancer Res 64(24): 8795-8799) etc. may be comprised in the modified adenoviruses of the invention.
  • the gene of interest located in (inserted into) the E3 or other suitable region of the adenoviral genome, is operably linked to a promoter element which is constitutively or inducibly active in the intended target cell (e.g., a cancer cell in a tumor to be treated).
  • a promoter element which is constitutively or inducibly active in the intended target cell (e.g., a cancer cell in a tumor to be treated).
  • Suitable promoters include, but are not limited to, the cytomegalovirus immediate early promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor- 1. alpha, promoter, the human ubiquitin c promoter, etc. (Colosimo et al. Biotechniques (2000) 29(2):314-318, 320-322, 324) and the PEG-3 promoter (U.S. Pat.
  • inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone); commercially available tetracycline-responsive or ecdysone-inducible promoters, etc.
  • the promoter may be selectively active in cancer cells, such as the prostate specific antigen gene promoter (O'Keefe et al. (2000) Prostate 45:149-157), the kallikrein 2 gene promoter (Xie et al. (2001) Human Gene Ther 12:549-561), the human alpha-fetoprotein gene promoter (Ido et al. (1995) Cancer Res 55:3105-3109), the c-erbB-2 gene promoter (Takakuwa et al. (1997) Jpn. J.
  • a PEG-3 promoter of the invention may also be a nucleic acid molecule that is at least about 85 percent, at least about 90 percent, or at least about 95 percent identical to SEQ ID NO : 1.
  • the promoter sequences may be full length or active fragments thereof that retain the PEG-3 expression pattern. In some cases, the fragments can be at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, and 850, base pairs of SEQ ID NO:1.
  • a virus of the invention expresses a chaperone protein, with or without mda-7 or other heterologous protein.
  • Chaperone proteins assist with the activity or function of other proteins in the cell. Many chaperone proteins are heat shock proteins or stress proteins, which prevent newly synthesized polypeptide chains and assembled subunits from aggregating. Chaperones can also assist in the assembly of nucleosomes from folded histones and DNA. Different stress proteins are very different in cellular functions and in their abilities to chaperone or bind antigens.
  • the virus expresses Grpl70.
  • Grpl70 is a newly characterized stress protein and the largest endoplasmic reticulum (ER)-resident molecular chaperone, and is a highly diverged relative of the hsp70 family. See, e.g., Park J, Easton DP, Chen X, MacDonald IJ, Wang XY, Subjeck JR.
  • Grpl70 has been shown to interact with transporter associated with antigen processing (TAP) translocated peptides and may be involved in polypeptide trafficking in the antigen presentation pathway.
  • TAP antigen processing
  • grpl70 is capable of interacting with specific receptors on professional antigen-presenting cells (APCs) and shuttling antigens into the endogenous presentation pathway efficiently, which results in antigen presentation and tumor-specific immunity.
  • APCs professional antigen-presenting cells
  • grpl70 also acts as a 'danger' signal that stimulates phenotypic and functional maturation of dendritic cells.
  • the chaperone protein aids in presenting the antigen from the cancer cell that has been killed by the virus to the immune system, thereby creating a "vaccine effect" against cancer cells of that particular type.
  • the immunochaperone grpl70 may simultaneously deliver both an adjuvant effect for activation of innate immunity and antigenicity the virus, which would dramatically increase the intrinsic immunogenicity of tumor killing.
  • tumor cells harbor a repertoire of unique, mutated-antigens as well as shared self-antigens
  • using the process of tumor cell death in combination with a potent immune activating agent/vaccine produced from within the same tumor cells may result in the permanent eradication of the primary tumor and metastases; thus preventing recurrence.
  • the chaperone protein is encoded by the virus to target the tumor and augments the immune system such that a robust tumor-specific immune response is provided, leading to a significantly improved control of distant and secondary tumors.
  • intratumoral co-administration of secretable grpl70 and the melanoma is provided by intratumoral co-administration of secretable grpl70 and the melanoma
  • differentiation-associated gene-7/interleukin-24 which is a cancer-specific and apoptosis- inducing gene, each encoded by a virus, effectively and markedly suppress treated local prostate tumors (see Example 2).
  • the tropism of the adenovirus may be altered to improve infection of adenovirus in target cells, e.g., cancer cells.
  • target cells e.g., cancer cells.
  • the absence of the primary adenoviral receptor, i.e. the Coxsackie-Adenovirus Receptor (CAR), in target cells is a substantial obstacle to effective gene therapy, as it limits the access of cells to therapeutic virus.
  • CAR Coxsackie-Adenovirus Receptor
  • adenoviral vectors may be targeted to alternative cellular receptors by genetically modifying surface properties of the viral capsid. Specific
  • modifications in the adenoviral capsid fiber can improve infectivity of prostate tumor cells by Triage viruses.
  • a modified adenovirus may further comprise a virion fiber or hexon capsid protein modification to facilitate infection of target cells and/or enhance targeting of an adenovirus vector to specific cell types.
  • viruses are referred to herein as "Triage Viruses”.
  • Triage Viruses Such capsid-modified adenoviruses are generically referred to in the literature as "infectivity enhanced" adenoviruses (Krasnykh et al. Cancer Res (2000)
  • one or more heterologous targeting ligands may be incorporated within the fiber. Based on the three dimensional model of the fiber knob, targeting ligand may be inserted into the HI loop of the fiber (Ruigork et al. (1990) MoI Biol 215:589-596). This loop is flexible, exposed outside the knob, is not involved in fiber trimerization, and its variable length is different among Ad serotypes suggesting that insertions or substitutions do not substantially affect fiber stability (Krasnyk et al. (1996) J Virol 70:6839-6846; Douglas et al. (1996) Nature Biotech 14:1574-1578).
  • two types of ligands may be introduced into the HI loop of the fiber: (i) the sequence coding for an RGD peptide, CDCRGRDCFC (SEQ ID NO:2), known to target tumors by binding with high affinity to several types of integrins thus facilitating binding via fiber-RGD/integrin interaction independent of the adenoviral CAR receptor (Krasnykh et al.
  • conditionally replicating adenoviral vector may be tropism-modified by altering the nature and properties of the hexon protein (Krasnyk et al. (1996) J Virol 70:6839-6846).
  • the hexon protein is in greater than twenty-fold abundance than the fiber protein.
  • the hexon protein may be modified to contain a small peptide ligand with high specificity for a cellular target.
  • hexon protein When expressed as a heterologous component of a hexon protein a small peptide ligand is presented on the surface of an adenovirus with high relative abundance. Peptide ligands when presented in this manner overcome potential lack of high affinity through increased avidity. Modification of hexon protein may be accomplished by genetic incorporation of DNA sequences coding for ligands into the hyper- variable regions of the hexon gene utilizing a suitable shuttle vector. In additional non- limiting embodiments, the fiber knob may be altered by genetic incorporation of alternate knob domains (Henry et al (1994) J Virol 68(6):5239-5246; Krasnyk et al. (1996) J Virol 70: 6839- 6846).
  • adenovirus having genetically modified infectivity tropisms may be used to provide enhanced infectivity and improved oncolytic potency.
  • the viruses are altered such that they bind to non-CAR receptors.
  • the tropism of the microbubble is changed by incorporating an agent with specific tropism into microbubbles.
  • microbubbles include a PSMA inhibitor, directed to the prostate specific membrane antigen (PSMA), to enhance the systemic targeting of the microbubbles to prostate cancer cells.
  • PSMA prostate specific membrane antigen
  • the methods of the invention may be used to target a variety of diseases and tissue types.
  • the invention can be used to treat any condition for which the virus suspended in microbubbles provides treatment, protection or amelioration.
  • the methods are used to target breast cancer, bronchial cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, hepatic carcinoma, urogenital cancer, ovarian cancer, testicular carcinoma, osteosarcoma, chondrosarcoma, gastric cancer, pancreatic cancer, nasopharyngeal cancer, thyroid cancer, neuroblastoma, astrocytoma, glioblastoma multiforme, melanoma, hemangiosarcoma, an epithelial cancer, a non-epithelial cancer such as squamous cell carcinoma, leukemia, lymphoma, and cervical cancer.
  • the methods may be used to target solid tumors or metastatic tumors.
  • the invention further provides a method for producing a cytopathic effect in a cell comprising administrering a virus in a microbubble suspension is administered, the virus is released at a desired location (e.g., by bursting the bubbles with ultrasound, and infecting the target cell(s) with a modified adenovirus according to the invention.
  • Types of cytopathic effects include a decrease in cell proliferation, a decrease in cell metabolism, and/or cell death.
  • the cell may be a cancer cell of for example, a nasopharyngeal tumor, a thyroid tumor, a central nervous system tumor (e.g., a neuroblastoma, astrocytoma, or glioblastoma multiforme), melanoma, a vascular tumor, a blood vessel tumor (e.g., a hemangioma, a hemangiosarcoma), an epithelial tumor, a non-epithelial tumor, a blood tumor, a leukemia, a lymphoma, a cervical cancer, a breast cancer, a lung cancer, a prostate cancer, a colon cancer, a hepatic carcinoma, a urogenital cancer, an ovarian cancer, a testicular carcinoma, an osteosarcoma, a chondrosarcoma, a gastric cancer, or a pancreatic cancer.
  • a cancer cell of for example, a nasopharyngeal tumor
  • the cell may be a cancer cell in a human or a non-human animal subject.
  • the amount of modified virus administered may be, but not by way of limitation, between about 1 x 10 10 to 1 x 10 13 pfu or at a multiplicity of infection (m.o.i.) of between about 10 and 5000 virus particles, between about 10 and 1000 virus particles, or between about 100 and 1000 virus particles, per estimated cell (where the tumor volume can be estimated, the number of cells in the tumor may be estimated (e.g., a spherical tumor having a diameter of 1 cm may be estimated to contain 10 9 cells; see James et al.
  • the effective amount may be administered in a series of inoculations, for example, between 1 and 15 inoculations, or between about 3 and 12 inoculations, or between about 3 and 7 inoculations, each containing between about 1 x 10 10 to 1 x 10 12 pfu or at a multiplicity of infection (m.o.i.) of between about 10 and 5000 virus particles, between about 10 and 1000 virus particles, or between about 100 and 1000 virus particles, per estimated cell.
  • inoculations for example, between 1 and 15 inoculations, or between about 3 and 12 inoculations, or between about 3 and 7 inoculations, each containing between about 1 x 10 10 to 1 x 10 12 pfu or at a multiplicity of infection (m.o.i.) of between about 10 and 5000 virus particles, between about 10 and 1000 virus particles, or between about 100 and 1000 virus particles, per estimated cell.
  • the mode of administration may be, but is not limited to, intra-tumor instillation, intravenous, intra-arterial, intrathecal, intramuscular, intradermal, subcutaneous, mucosal via pulmonary or other route, direct nasal installation, etc.
  • the invention provides for methods of using the modified adenoviral vectors of the invention to treat forms of cancer which are refractory to conventional therapies ("refractory cancers"), or to inhibit the proliferation of a cell of a refractory cancer, or to inhibit tumor growth and/or metastasis of a refractory cancer.
  • refractory cancers conventional therapies
  • a cancer which has not shown adequate clinical response to a treatment agent, or combination thereof, which is not a modified adenoviral vector of the invention is considered such a refractory cancer.
  • a cancer which overexpresses as antiapoptotic protein such as but not limited to Bcl-2 or BcI- xL
  • a refractory cancer is apoptosis-resistant and/or treatment resistant prostate cancer.
  • a breast cancer which overexpresses Bcl-2, a small-cell lung cancer which overexpresses Bcl-2, a non-small cell lung cancer which overexpresses Bcl-2, and a liver cancer which overexpresses Bcl-2 are each considered to be refractory cancers.
  • the invention provides for a method of treating a subject suffering from a cancer, where the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, hepatic carcinoma, urogenital cancer, ovarian cancer, testicular carcinoma, osteosarcoma, chondrosarcoma, gastric cancer, pancreatic cancer, nasopharyngeal cancer, thyroid cancer, neuroblastoma, astrocytoma, glioblastoma multiforme, melanoma, hemangiosarcoma, an epithelial cancer, a non-epithelial cancer such as squamous cell carcinoma, leukemia, lymphoma, and cervical cancer, comprising administering, the subject, an effective amount of a modified adenovirus according to the invention.
  • the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, hepatic carcinoma, urogenital cancer, ovarian cancer, testi
  • an effective amount of modified adenovirus may be between about 1 x 10 10 to 1 x 10 12 pfu or a multiplicity of infection (m.o.i.) of between about 10 and 5000 virus particles, between about 10 and 1000 virus particles, or between about 100 and 1000 virus particles, per estimated cell, and the effective amount may be administered in a series of inoculations, for example, between 1 and 15 inoculations, or between about 3 and 12 inoculations, or between about 3 and 7 inoculations, each containing between about 1 x 10 10 to 1 x 10 12 pfu or at a multiplicity of infection (m.o.i.) of between about 10 and 5000 virus particles, between about 10 and 1000 virus particles, or between about 100 and 1000 virus particles, per estimated cell.
  • m.o.i. multiplicity of infection
  • Treating a subject suffering from a cancer means one or more of the following: decreasing tumor volume; decreasing rate of tumor growth; increasing survival; decreasing tumor grade; inhibiting metastasis (meaning inhibiting dissemination and/or
  • metastatic cells growth/proliferation of metastatic cells
  • increasing time of survival increasing time of survival
  • improving quality of life e.g., decreasing pain, increasing ability to perform activities.
  • the invention in further non-limiting embodiments provides for a method of treatment of various types of cancer cells involving combined treatment with a microbubble suspension of a Terminator or Triage Virus in combination with radio- or chemotherapeutic agents.
  • PEG-3 promoter activity is enhanced by DNA damaging agents and ionizing radiation (Su et al. (1999) Proc Natl Acad Sci USA 96(26): 15115-151120; Su et al. (2002) J Cell Physiol 192(l):34-44). Therefore enhanced viral replication leading to enhanced cytolysis of tumor cells may be achieved.
  • Combination therapy includes but is not limited to simultaneous or serial treatment with a Terminator or Triage Virus embodied in instant invention and standard radiotherapy or chemotherapy regimes.
  • Chemotherapy may include but is not limited to treatment with appropriate doses of chemotherapy agents such as Cisplatin, Adriamycin, Doxorubicin, Paclitaxel or other Taxol derivatives, etc.
  • chemotherapy agents such as Cisplatin, Adriamycin, Doxorubicin, Paclitaxel or other Taxol derivatives, etc.
  • specific targeting to an organ, tumor or tissue type or enhanced infectivity is obtained by utilizing an appropriate Triage Virus.
  • a combination of two or more Terminator or Triage Viruses suspended in microbubbles may be used for a method of treatment of a cancer or other disease state.
  • two or more Terminator or Triage Viruses expressing distinct genes of interest may be used in combination (administered concurrently or sequentially) for treatment in a human or non-human animal subject.
  • Non- limiting examples of such combinations include treatment of a subject with two Terminator viruses, one expressing a gene of interest encoding IFN-alpha, IFN-beta, IFN-gamma, IL-2, IL-4, IL- 12, RIG-I, mda-5 etc. and the other expressing a gene of interest encoding a tumor specific antigen or an immune accessory molecule such as Carcino-Embryonal Antigen (CEA), the B7.1 gene, lymphocyte homing receptor or HLA antigen gene.
  • CEA Carcino-Embryonal Antigen
  • Terminator or Triage Viruses expressing appropriate genes of interest may also be utilized to restore or boost the responsiveness of a subject to a specific form of conventional radio-, chemo- or immunotherapy.
  • Non-limiting examples of such viruses contain a gene of interest which encodes the EGFR (Epidermal Growth Factor Receptor) or related variants such as the Her-2/neu receptor thereby enhancing a subject's responsiveness to therapies such as Herceptin in breast cancer patients or other anti-EGRF therapies such as Gefitinib (Iressa, ZD 1839) an EGFR specific tyrosine kinase inhibitor or the tyrosine kinase inhibitor NVP-AEE788 (AEE788) which blocks both the EGF and VEGF signaling pathways.
  • EGFR Epidermatitis
  • Viruses containing a gene if interest encoding the androgen receptor (AR) may be used to enhance or restore responsiveness to anti-androgen therapy in androgen refractive forms of prostate cancer.
  • Triage Viruses that target expression to specific tissues such as breast or prostate and in addition, restore responsive therapeutic targets such as EGFR or AR may be utilized to localize and enhance the efficiency of a particular form of radio-, chemo- or immunotherapy.
  • the methods of the invention include systemic delivery of virus via microbubble suspensions. Once the suspension is administered the microbubbles can be disrupted locally to releasing virus at a desired location. In one embodiment, the microbubbles are disrupted using ultrasound. Without being bound by theory, it is believed that ultrasound-targeted microbubble destruction (UTMD) enables focal release of entrapped materials as well as the creation of small shock waves that increase cellular permeability.
  • UTMD ultrasound-targeted microbubble destruction
  • Gas filled microbubbles have been conventionally used as contrast agents for diagnostic ultrasound. They have also been described for therapeutic applications, such as enhancement of drug penetration (Tachibana et al., U.S. Pat. No. 5,315,998), as thrombolytics (e.g. Porter, U.S. Pat. No. 5,648,098), and for drug delivery. Reports of use of microbubbles for drug delivery have generally described the use of some external method of releasing the drug from the microbubbles at the site of delivery, by, for example, raising the temperature to induce a phase change (Unger, U.S. Pat. No. 6,143,276) or exposing the microbubbles to ultrasound (Unger, U.S. Pat. No. 6,143,276; Klaveness et al., U.S. Pat. No. 6,261,537; Lindler et al., Echocardiography 18(4):329, May 2001, and Unger et al.,
  • a major challenge for effective gene therapy is the ability to specifically deliver nucleic acids and potentially toxic gene products directly into diseased tissue.
  • Progress in gene therapy has been hampered by concerns over the safety and practicality of viral vectors, particularly for intravenous delivery, and the inefficiency of currently available non- viral transfection techniques [12].
  • Viruses are appealing delivery vectors because of their ability to efficiently transfer genes with sustained and robust expression.
  • Recombinant Ads are one of the most common gene transfer vectors utilized in human clinical trials, but systemic administration of this virus is thwarted by host innate and adaptive antiviral immune responses which can limit and/or preclude repetitive treatment regiments [13].
  • US contrast agents microbubbles
  • MB Microbubbles
  • the ideal MB diameter most likely is between 2.5 to 4 ⁇ m. This is small enough to prevent entrapment within the pulmonary capillary bed (ranging from 5 to 8 ⁇ m in diameter), but big enough to entrap and protect viral vectors such as Ad from the environment.
  • microspheres effectively lower the energy threshold for non-thermal cavitation. This allows diagnostic transducers operating within the energy levels mandated by the FDA to be used for drug/gene delivery.
  • Ultrasound-targeted microbubble destruction UTMD
  • UTMD Ultrasound-targeted microbubble destruction
  • the microbubbles protect the viruses from rapid degradation by the immune system, thus allowing for intravenous injection rather than direct target organ delivery by catheter-based approaches or operative bed injection [12, 17]. This is particularly important in cancer gene therapy of potentially inaccessible tumors because the microbubbles may also limit the amount of inflammatory response to the viruses and may allow repeated injections.
  • Targeson' s agents are lipid-encapsulated perfluorocarbon microbubbles with a mean diameter of 2.5 ⁇ m that can be used in a wide variety of animal models, and are compatible with virtually all ultrasound scanners [20].
  • Targeson agents are normally sold as already reconstituted contrast agents that are stable for three months from arrival, and for this study we obtained a custom made freeze-dried Targeson contrast agent (perfluorocarbon microbubbles, encapsulated by a lipid monolayer and poly(ethyleneglycol) stabilizer) to be reconstituted with the viruses as previously described [12].
  • FIG. IA A portable SonoSite Micro-Maxx ultrasound platform (SonoSite, Inc., Bothell, WA) equipped with a L25 linear array transducer set at 0.7 Mechanical Index (MI), 1.8 MPa for 10 min was used to sonoporate only the tumor implanted on the right side ( Figure IA). Mice were sacrificed 96 hr after treatment and tumors (right and left side), lung, heart, liver and kidney were harvested and snap frozen. Figure IB shows the specific delivery to the right tumor as evidenced by expression of the green fluorescence protein (GFP) in an immunoblot in which total protein extracts were run on a 10% SDS-PAGE. As a GFP control, we ran a GST-GFP fusion protein. Protein gel loading was normalized using ⁇ -actin as a control.
  • GFP green fluorescence protein
  • US-targeted microbubble destruction enables focal release of entrapped materials as well as the creation of small shock waves that are visualized as an enhancement of the image on the US scanner.
  • panel A depicts the B-mode US imaging of a sonoporated tumor before injection with the microbubble/ Ad-GFP complex contrast agent.
  • panel B shows the B-mode ultrasound imaging of the same sonoporated tumor following microbubble/ Ad-GFP complex injection.
  • the image enhancement of the targeted tumor from cavitation of the microbubbles within the US field of view is clearly discernable indicating that the US settings are efficient in targeting microbubble destruction.
  • Microbubble assisted Ad.mda-7 gene delivery inhibits DU-145 human prostate cancer growth in vivo.
  • Ad.mda-7 potently suppresses the growth of human cancer cells with no apparent toxicity to normal cells [6, 8, 9, 21-30].
  • Repeated intratumoral administration of Ad.mda-7 to tumor xenografts of various histological origin results in growth suppression via induction of apoptosis and anti-angiogenic mechanisms [4-9, 25, 28, 31].
  • mda-7/ ⁇ L24 induces a profound "bystander" antitumor effect resulting in tumor growth suppression not only in the treated tumors, but also in untreated distant tumors [6, 8-11, 22, 23, 25, 27, 32- 36]. Although these results have been encouraging, this approach is limited since systemic delivery of Ad for treatment of disseminated cancer have not shown significant efficacy.
  • DU-145 human prostate carcinoma cells and DU-145 cells genetically engineered to express elevated levels of BCI-X L (DU-Bcl-xL) [4], which is a common event in advanced prostate cancer and provokes resistance to multiple chemotherapeutic agents and to m ⁇ i ⁇ -7/IL-24 [2-5].
  • BCI-X L DU-Bcl-xL
  • the therapeutic arm of this work included two different viral constructs to deliver m ⁇ i ⁇ -7/IL-24, Ad.mda-7, a nonreplicating Ad similar to the one used in Phase I clinical trials [26], and the CTV, a conditionally replication competent Ad capable of expressing mda-7/IL-24 that has been previously shown to completely eradicate not only primary breast, prostate and melanoma tumors but also distant tumors by intratumoral injections in a nude mouse model [11, 33, 34, 37]. [0100] To test this new therapeutic approach for tumor delivery, DU- 145 or DU-Bcl-xL tumor xenografts were established on both flanks of nude mice by injecting 2 x 10 6 cells in each side of the animals.
  • MI Mechanical Index
  • Microbubble assisted CTV gene delivery eradicates prostate cancer growth in vivo.
  • further experiments were performed in nude mice. Tumor xenografts were again established on both flanks of nude mice by injecting 1.5 x 10 6 DU-145 or DU-Bcl-xL cells. After palpable tumors developed in 5 weeks (25-50 mm ⁇ 3), four injections of the various microbubble/ Ad complexes into the tail vein once a week (for a total of four weeks) with US for 10 min on the tumor on the right side were performed. No treatment was performed on the tumor xenografted on the left flank.
  • Control tumors treated with Ad-GFP-microbubble complexes were mostly TUNEL negative, showing a few cells positive to the TUNEL colorimetric reaction as depicted in Figure 5A (sonoporated tumor on the right flank) and Figure 5B (untreated tumor). Additionally, DU- 145 tumor xenografts treated with Ad.m ⁇ i ⁇ -7-microbubble complexes showed a higher percentage of TUNEL positive tumor cells (Figure 5C) than the untreated left flank ( Figure 5D).
  • mice with established xenografts on both flanks were injected in the tail vein with the microbubble/Ads complexes, and only the tumors on the right flank were sonoporated.
  • Animals receiving the Ad-GFP-microbubble complexes plus US treatment showed no statistically significant effect on the growth of DU-Bcl-xL tumors ( Figures 6A and B).
  • the data presented is the average tumor volumes of 9 mice receiving therapeutic gene treatment and 6 mice receiving Ad-GFP treatment.
  • FIG. 9A shows the ultrasound image and measurements of a DU- 145 tumor before treatment with Ad.m ⁇ i ⁇ -7-microbubble complexes.
  • Panels B and C demonstrate the volume reduction in the same tumor after 2 and 4 weeks of treatments with Ad.m ⁇ i ⁇ -7-microbubble complexes and US.
  • Figure 9D shows the B-mode scan image and measurements of a DU-Bcl-xL tumor before treatment with C7T-microbubble complexes.
  • Panels E and F emphasize the dramatic volume reduction in the same tumor after 2 and 4 wks of treatments with C7T-microbubble complexes and US leading to the eradication of the tumor xenograft. Additionally, no tumor regrowth in the primary or distant sites was evident C7T-microbubble complex and US-treated DU-Bcl-xL animals after an additional three weeks post-treatment. To investigate if the tumor would reappear after a longer period of time following the last treatment, three out often animals initially treated with CTV- microbubble complexes were not sacrificed at the endpoint of the study and were maintained for an additional 3 months. The mice were then sacrificed and dissected to look for potential tumor recurrence and/or eventual tumor spread.
  • MicroMaxx (SonoSite, Inc., Bothell, WA) after a simple intravenous injection.
  • prostate cancer is commonly a relatively slow-growing disease, it may be necessary to use repeated gene therapy applications, with single or multiple genes, over the life span of the patient.
  • gene therapy protocols that delimit virus exposure to the immune system and can be administered multiple times during a patient's lifetime are appealing. This possibility will need to be explored in the future using tumor-bearing immune competent animals.
  • Ad.mda-7 a replication incompetent adenovirus
  • Ad.PEG-ElA-m ⁇ i ⁇ -7 conditionally replication competent Ad
  • CRCA Conditionally replication competent Ads
  • the novel C7T CRCA that employs the progression elevated gene-3 (PEG-3) promoter that functions in all types of cancer cells [11, 34, 37, 45, 46], irrespective of their p53 or Rb/retinoblastoma gene status, with very limited to no activity in normal cells has been constructed.
  • CTV cancer terminator virus
  • Ad replication through the ElA gene is driven by the cancer- specific promoter of progression elevated gene- 3 [PEG-V) [47], which results in concomitant production of mda-7/IL-24 from the E3 region of the Ad.
  • This C7Y generates large quantities of MDA-7/IL-24 as a function of Ad replication uniquely in cancer cells that not only has cancer-selective apoptosis-inducing properties but also displays a plethora of indirect antitumor "bystander” activities, including distant tumor growth suppression and apoptosis, immune modulation and anti-angiogenesis [6-9, 22, 32, 35, 37, 41].
  • a limiting factor in effective gene therapy when employing intravenous viral delivery and when using CRCA is the effect of the immune system in neutralizing Ads [13].
  • a means of shielding the initial viral delivery vector using microbubbles in principle permits enhanced delivery of the viral payload to tumors when coupled with US [12].
  • mda-7/IL-24 can directly induce apoptosis when expressed inside cancer cells and can also induce growth suppression, apoptosis and endogenous MDA-7/IL-24 protein expression and secretion when added as a purified protein through interactions with the IL-20R1/IL-20R2 and IL-22R1/IL- 20R2 cell surface receptors [6, 8, 32, 35].
  • MDA-7/IL-24 As a secreted cytokine, MDA-7/IL-24 also induces an array of potent immunomodulatory proteins from immune cells, including IL-6, IFN- ⁇ , tumor necrosis factor- ⁇ , IL- l ⁇ , IL- 12, and granulocyte macrophage colony-stimulating factor [32]. These cytokines secreted by peripheral blood mononuclear cells can activate antigen- presenting cells to present tumor antigens, thereby triggering an antitumor immune response [48]. These observations have been recapitulated in a Phase I clinical trial involving intratumoral injection of Ad.mda-7 (INGN 241) in patients with advanced carcinomas and melanomas [8, 21, 28]. In principle, the 'bystander" effects elicited by MDA-7/IL-24 are concentration dependent [35] and large amounts of this cytokine generated by the CTV would be predicted to have an enhanced therapeutic impact in the patient. Moreover, the 'bystander" effects elicited by M
  • the DU-145 (human prostate adenocarcinoma), cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and the DU-Bcl-xL cell line, which constitutively expresses elevated levels of BCI-X L has been described previously [4].
  • the cell lines were grown at 37 0 C, in a 5% CO2/95% atmosphere, in Dulbecco's modified Eagle's medium (Mediatech Inc., Herndon, VA) supplemented with 10% fetal bovine serum (FBS) from Hyclone, Inc., (Logan, UT).
  • Dulbecco's modified Eagle's medium Mediatech Inc., Herndon, VA
  • FBS fetal bovine serum
  • Ad- GFP which expresses the green fluorescence protein gene under the strong cytomegalovirus (CMV) constitutive promoter was generated using the AdEasy system (Carlsbad, CA); the conditionally replication competent cancer terminator virus C7Y (Ad.PEG-ElA-m ⁇ i ⁇ -7) [11, 34, 37] and Ad.mda-7 [26] were amplified and purified with the BD Adeno-X virus purification kit (BD Biosciences, Mountain View, CA) following manufacturer's directions. Viral titers were determined by a plaque assay and the titer was adjusted to 1.2 x 10 12 plaque- forming units (pfu)/mL as described [26].
  • CMV cytomegalovirus
  • Microbubbles were reconstituted in the presence or absence of 1 mL of 1.2 x 10 12 pfu of Ads and unenclosed, surface associated Ads were inactivated as previously described [12].
  • US exposure was achieved with a Micro- Maxx SonoSite (SonoSite, Bothell, WA) US machine equipped with the transducer L25 set at 0.7 Mechanical Index (MI), 1.8 MPa for 10 min.
  • MI Mechanical Index
  • mice were sacrificed and fresh tumor (right and left flank), heart, lung, liver, and kidney tissues were harvested and snap frozen in liquid nitrogen.
  • Mice receiving mda-7/IL-24 gene-microbubble US guided therapy were sacrificed at the endpoint of the study (5-6 wks after gene therapy injections).
  • Tissues were homogenized and equal amounts of proteins were run on a SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then incubated with the monoclonal anti-GFP 1 :2,000 for 1 hr at room temperature and then washed three times in TBS-T.
  • Monoclonal anti-MDA-7/IL-24 (GenHunter, Inc, Nashville, TN) was incubated 1 :2,000 for 1 hr at room temperature and then washed three times in TBS-T.
  • Monoclonal anti ⁇ -actin (1 :5,000) was incubated 1 hr at room temperature and then washed three times in TBS-T.
  • Appropriate secondary HRP-conjugated antibodies 1 :20,000 were incubated 45 min at room temperature and washed three times with TBS-T. Signals were developed on an X-ray film after reaction with an Electrogenerated ChemiLuminescence (ECL) Supersignal kit (Pierce, Rockford, IL).
  • mice Animal study and ultrasonic bubble destruction. Animal studies were performed in accordance with NIH recommendations and the approval of the institutional animal research committee. Animal care and humane use and treatment of mice were in strict compliance with (1) institutional guidelines, (2) the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, Washington, DC, 1996), and (3) the Association for
  • mice congenitally athymic BALB/c nude mice, homozygous for the nu/nu allele, bred in our laboratory.
  • the colony of the mice was developed from breeding stock obtained from Charles Rivers Laboratories, Wilmington, MA. The mice were maintained in isolation in autoclaved cages with polyester fiber filter covers, under germ-free conditions; all food, water, and bedding were sterilized.
  • mice were sedated in an IMPAQ6 anesthesia apparatus (VetEquip Inc, Pleasanton, CA) that was saturated with 3-5% Isofluorane and 10-15% oxygen with the aid of a precision vaporizer (VetEquip Inc, Pleasanton, CA) to deliver the appropriate amount of anesthetic and to induce anesthesia.
  • the mice were placed on a warmed mat with 37 0 C circulating water for the entire procedure.
  • A27-gauge needle with a heparin lock was placed within a lateral tail vein for administration of contrast material.
  • the nude mice received injections of 100 ⁇ L of microbubbles with/without Ads through the tail vein for 5 wks/once a wk.
  • mice were split into two control groups (one control group receiving 100 ⁇ L of microbubbles and US, and another control group receiving both microbubbles/ Ad-GFP and US) and eight active groups of 10 mice each (all receiving microbubbles and Ad.mda-7 or CTV and US).
  • Six additional control groups were set up which received direct i.v. injections of 1 OO ⁇ L of the Ads (Ad-GFP, Ad.mda 7/IL-24, or CTV) in the presence or not of US .
  • mice were humanely sacrificed by placing them in a CO 2 gas jar placed in a ventilated fume hood.
  • the tumors (right and left flank), heart, lungs, kidneys, and liver were harvested.
  • Tissues to be sectioned were dry snap frozen or placed either in OCT (Sakura Finetek USA, Inc., Torrance, CA), frozen in liquid nitrogen, and stored at -8O 0 C or were preserved in neutral buffered formalin at 4 0 C prior to embedding in paraffin for immunohistochemical analysis.
  • TUNEL assay Determination of apoptotic cells by TUNEL assay.
  • a TUNEL method was used for the detection of apoptotic cells.
  • Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24. Oncogene 22: 8758-8773.
  • MDA-7 interleukin-24 inhibits the proliferation of renal carcinoma cells and interacts with free radicals to promote cell death and loss of reproductive capacity.
  • Adenovirus-mediated mda-1 (IL24) gene therapy suppresses angiogenesis and sensitizes NSCLC xenograft tumors to radiation.
  • Kirn, DH (1997). ONYX-015, an ElB gene-attenuated adenovirus, causes tumor- specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents. Nat Med 3: 639-645. 45. Sarkar, D, et al. (2005). Targeted virus replication plus immunotherapy eradicates primary and distant pancreatic tumors in nude mice. Cancer Res 65: 9056-9063.
  • mda-7 Melanoma differentiation associated gene-7
  • IL-24 interleukin-24
  • m ⁇ i ⁇ -7/IL-24 does not induce growth suppressive or toxic effects in normal cells (3-5).
  • m ⁇ i ⁇ -7/IL-24 is also capable of regulating cell cycle (6), inhibiting angiogenesis (7), sensitizing cancer cells to radiation therapy (8, 9).
  • Recent studies highlighted a potent 'bystander' antitumor activity exhibited by mda-7/IL-24 in adjacent tumor cells (10), which may provide a means of obviating the technical difficulty of transducing the entire tumor mass with this gene.
  • apoptotic cells are largely ignored by the immune system (15), or may even induce tolerance (16). Only 'inflammatory' or 'immunogenic' cell killing, which is distinguished from normal homeostatic processes, can be sensed by the host immune system and is able to trigger the activation of immune components (17-19).
  • Grpl70 has been shown to interact with transporter associated with antigen processing (TAP) translocated peptides and may be involved in polypeptide trafficking in the antigen presentation pathway (21, 22).
  • TAP antigen processing
  • grpl70 is capable of interacting with specific receptors on professional antigen-presenting cells (APCs) and shuttling antigens into the endogenous presentation pathway efficiently, which results in antigen presentation and tumor- specific immunity (23, 24).
  • APCs professional antigen-presenting cells
  • Tumor-derived grpl70 or grpl70 complexed with tumor-associated antigens are highly effective in eliciting potent antigen and tumor specific antitumor immune responses in various murine tumor models (23, 25).
  • grpl70 In addition to promoting antigen cross-presentation, grpl70 also acts as a 'danger' signal that stimulates phenotypic and functional maturation of DCs, as indicated by up- regulation of MHC class II and co-stimulatory molecules, secretion of proinflammatory cytokines and chemokines (26). More recently, we demonstrated that extracellular targeting of grpl70 by molecular engineering strongly enhanced the immunogenicity of a poorly immunogenic tumor in vivo (27).
  • mice and Cell lines 8 to 12-week-old male C57BL/6 mice purchased from the National Institutes of Health animal facilities were maintained in a pathogen- free facility at Roswell Park Cancer Institute. Animal care and experiments were approved by the
  • TRAMP-C2 cell line was derived from a prostate tumor that arose in a TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mouse in the C57BL/6 background (28).
  • TRAMP-C2 cells, C2 cells transduced with OVA (C2-OVA) and B16 melanoma cells are maintained in DMEM containing 10% fetal bovine serum, 2mM L-glutamine, and 100U/ml penicillin/streptomycin.
  • Adenovirus construction and characterization The recombinant replication- defective Ad.mda-7 virus was created in two steps as described previously (3).
  • the adenovirus carrying a secretable form of the grpl70 gene (Ad.sgrpl70) was constructed using BD Adeno-XTM Adenoviral Expression System (BD Bioscience, Palo Alto, CA). To distinguish the secretable grpl70 from endogenous grpl70, a His-tag was fused to the C- terminus of mouse grpl70, in which the KNDEL endoplasmic reticulum signal has been eliminated (27). This modified cDNA was inserted into the Nhe I/Xba I cloning sites of the pShuttle 2 plasmid, and subsequently cloned into I-Ceu I/PI-Sce I sites of Adenoviral vector.
  • adenoviral vectors were produced in HEK293 cells and infection titers were determined by plaque tittering on 293 cells. Viruses were concentrated and purified using AdenoPACK Maxi columns (Sartorius Stedim Biotech) according to the procedure provided by the manufacturer. Endotoxin levels are determined by using a chromogenic limulus amebocyte lysate kinetic assay kit (Kinetic-QCL; Biowhittaker, Walkersville, MD). Cells were infected with viruses with different multiplicity of infection (MOI) under standard culture conditions. Expression of the secreted grpl70 in the supernatants of the infected cells was examined using antibodies against grpl70 and His-tag as previously described (27). Expression o ⁇ mda- 7/IL-24 was examined by immunoblotting using murine anti-m ⁇ i ⁇ -7/IL-24 monoclonal antibodies (Gene Hunter, Inc).
  • Annexin V binding assays were used to determine apoptosis induction as previous described (29). Briefly, 24 h after virus infection at a MOI of 300 plaque-forming units (pfu) per cell, cells were harvested, washed and resuspended in binding buffer containing 2 mM CaCl 2 . Cells were stained with FITC-labeled Annexin V (BD Biosciences, Palo Alto, CA) and propidium iodide for 15 min at room temperature, and analyzed by Flow cytometry. In addition, cells were subjected to immunoblotting analysis using antibodies for Poly (ADP-ribose) polymerase (PARP) (Santa Cruz Biotech).
  • PARP Poly (ADP-ribose) polymerase
  • mice were randomly divided into five groups and received Ad.GFP, Ad.mda-7, Ad.sgrpl70, or Ad.mda-7 plus Ad.sgrpl70.
  • Viruses were administrated intratumorally in 50 ⁇ l PBS (5 x 10 8 pfu per mouse).
  • PBS 5 x 10 8 pfu per mouse
  • All treatments were given every other day for a total of 4 doses.
  • Tumor growth is monitored by measuring perpendicular tumor diameters using an electronic digital caliper. To determine the effect of the combined therapies on distant tumors, mice were established with tumors in both flanks.
  • CD4 + , CD8 + T-cell subsets were delivered into tumors in the left flank only. Growth of contralateral tumors was followed to determine systemic antitumor immunity. Depletion of CD4 + , CD8 + T-cell subsets was accomplished by i.p. injection of 200 ⁇ g GKl .5 and 2.43 mAb respectively as previously described (27). Effective depletion of cell subsets was maintained by the antibody injections once a week for the duration of
  • ELISPOT Enzyme-linked immunosorbent spot
  • CTL assays Splenocytes were isolated from immunized mice two weeks after immunization and stimulated with 1 ⁇ g/ml H-2K b restricted CTL epitope OVA 257 ⁇ (SIINFEKL) or mitomycin C-treated C2 tumor cells to determine antigen-specific, IFN- ⁇ secreting T-cells as previously described (30).
  • splenocytes were stimulated with mitomycin C-treated tumor cells or 1 ⁇ M OVA 2 5 7 -264 in the presence of IL-2 (20 U/ml) for 6 days.
  • CD8 + T-cells were used as effector cells in a chromium release assay as described (30).
  • the modified grpl70 should be secreted after protein synthesis.
  • the His-tag was fused to the COOH terminus of the sgrpl70 gene.
  • the fusion gene of sgrpl70-His was inserted into El/E3-deleted adenovirus- based vectors under the control of CMV promoter for constitutive and effective gene expression (i.e., Ad.sgrpl70).
  • the replication-defective virus vectors encoding this new fusion gene were successfully packaged and expanded in HEK293 cells.
  • TRAMP-C2 cell line that was established from the spontaneous tumor of the autochthonous transgenic adenocarcinoma of mouse prostate (TRAMP) model (28).
  • TRAMP-C2 cells form slowly growing, vascularized and poorly immunogenic tumors.
  • Ad.sgrpl70 at different MOIs
  • expression of the secretable grpl70 gene was examined in the supernatants of the infected cells. Robust expression of the sgrpl70 gene could be observed from day 1 at a MOI of 100 (Fig. 12B).
  • Adenovirus-mediated mda-7/lL-24 expression inhibits TRAMP-C2 tumor cell growth by inducing tumor apoptosis.
  • adenovirus-mediated mda-7/ ⁇ L-24 expression could induce growth suppression and apoptosis in TRAMP-C2 tumor cells (Fig. 13).
  • Immunoblotting analysis confirmed the expression of m ⁇ i ⁇ -7/IL-24 gene in C2 cells infected with Ad.mda-7 at different MOIs (Fig. 13A, upper).
  • Ad.mda-7 generated multiple bands because of glycosylation, ranging in size from 20 to 30 kDa.
  • Annexin V staining followed by fluorescent-activated cell sorting analysis was carried out to determine early apoptotic changes in C2 cells after infection with 300 pfu/cell of Ad.GFP or Ad.mda-7 (Fig. 13B). Whereas significant increase in apoptotic cells were observed in C2 tumor cells following Ad.mda-7 infection, no such change was evident in C2 cells treated with Ad.GFP or left untreated. In addition, cleavage of PARP was detected only in C2 cells infected with Ad.mda-7 (Fig. 13C), suggesting that overexpression of mda-7/IL-24 in mouse tumor cells induces apoptosis in an manner similar to that observed in human cancer cells (31).
  • Ad.mda-7 displays similar cancer-specific growth-suppressive and apoptosis-inducing properties in TRAMP-C2 prostate cancer cells, with no toxic effects in normal cells.
  • Ad.mda-7, Ad.sgrpl70 or Ad.mda-7 plus Ad.sgrpl70 total 5 X 10 8 pfu per injection
  • Fig. 14A It was observed that administration of Ad.GFP or PBS (data not shown) had little effect on C2 tumors, whereas treatment with either Ad.mda-7 or Ad.sgrpl70 significantly delayed tumor growth.
  • treatment with Ad.mda-7 combined with Ad.sgrpl70 exhibited much more potent tumor-suppressive activities (Fig. 14B). Additionally, tumors in 20% of mice treated with Ad.mda-7 plus Ad.sgrpl70 showed complete and prolonged regression.
  • mice receiving Ad.mda-7 plus Ad.sgrpl70 showed a significant inhibition in tumor growth on the untreated, contralateral flank, whereas in mice injected with Ad.mda-7, growth of contralateral tumors was essentially unimpeded.
  • mice treated with Ad.mda-7 plus Ad.sgrpl70 were protected from rechallenge with C2 tumor, whereas those treated with Ad.GFP or Ad.mda-7 alone were still susceptible. It was seen that Ad.sgrpl70 treatment failed to protect mice from rechallenge with the same tumor, suggesting that the m ⁇ i ⁇ -7/IL-24-mediated tumor apoptosis plays an important role in induction of antitumor responses.
  • mice established with C2-OVA tumors were treated with Ad.mda-7, Ad.sgrp 170, or Ad.mda-7 plus Ad.sgrp 170.
  • Splenocytes were isolated from the treated mice one or three weeks following the last injection.
  • the ELISPOT assay was used to examine the OVA-specific IFN- ⁇ production by splenocytes upon stimulation with MHC I-restricted CTL epitope for OVA, i.e., OVA257-264 (SIINFEKL) (Fig. 15B, top).
  • ELISPOT assay was performed to measure the tumor-specific secretion of IL-4 by splenocytes from the treated animals. It was observed that splenocytes of Ad.mda-7 treated mice produced higher levels of IL-4 compared to cells from animals treated with Ad.sgrp 170 or Ad.mda-7 plus Ad.sgrp 170 (Fig.15C). Effector CD8 + -T cell function, i.e., cytolytic activity, was assessed by chromium release assay using C2-OVA tumor cell as a target (Fig. 15D).
  • CD8 + T-cells are primarily involved in the systemic antitumor effects provided by the combined gene therapies.
  • C2-OVA tumor-bearing mice were depleted of CD4 + or CD8 + T-cell subsets by antibody injections prior to the initiation of treatment (Fig. 16A). It was found that depletion of CD8 + T-cell abrogated the therapeutic efficacy of the combined treatments, indicating that CD8 + T-cell plays a critical role in tumor eradiation.
  • antitumor immunity remained intact in mice depleted of CD4 + T-cell or those treated with control IgG.
  • Ad.mda-7 and Ad.sgrpl70 are capable of inducing antitumor immunity.
  • a modified treatment protocol was used to deliver Ad.mda-7 and Ad.sgrpl70 at different time points as described in Fig. 17A.
  • significant enhancement of m ⁇ i ⁇ -7/IL-24-targeted therapy by sgrpl70 was observed in mice receiving the two therapeutic agents either together (T) or separately (S) (Fig.
  • cytolytic activity assays showed that concurrent delivery of Ad.mda-7 and Ad.sgrpl70 at the same time promoted a more potent CTL response than separate administration of Ad.mda-7 and Ad.sgrpl70 (Fig. 17D).
  • grpl70 Two features make grpl70 a highly potent, "physiological" mammalian adjuvant that can be used for active immunotherapy: a cross-priming carrier and activator of innate immunity (25, 26). Based on our recent report demonstrating that extracellular targeting of grpl70 dramatically improves the immunogenicity of poorly immunogenic tumors, including melanoma (27) and prostate cancer (Gao et al., unpublished data), we postulate that tumor- specific killing by adenovirus-mediated mda-7/lL-24 expression, taken together with simultaneous release of tumor-derived grpl70, will provide both 'danger' signals and tumor- associated antigens to APCs (e.g., DCs), leading to strong tumor-specific immunity.
  • APCs e.g., DCs
  • splenocytes from Ad.m ⁇ i ⁇ -7-treated mice consistently produced higher levels of IL-4 than those from animals treated with Ad.sgrpl70 or Ad.mda-7 plus Ad.sgrpl70 (Fig. 15C), which lends support to an early study in which murine mda-1, also called IL-4-induced secreted protein (FISP), was postulated to be a type 2 cytokine (33).
  • FISP murine mda-1
  • FISP IL-4-induced secreted protein
  • the intratumoral immunotherapy drastically reduces the possibility of tumor escape due to antigen loss or tumor heterogeneity, since the approach uses the tumor against itself and grpl70 derived from tumor cells is directed against a diverse antigenic repertoire.
  • the efficacy of which is strictly limited by the quantity of stress proteins and the availability of tumor specimens the mda-7/ ⁇ L-24 and sgrpl70-based therapy described here is universally applicable and more cost effective, since the vaccine is generated at the site of the patient's own tumor using his own tumor antigens.
  • m ⁇ i ⁇ -7/IL-24-based approach should display enhanced safety in the clinic because of the cancer specificity of mda-7/lL-24 and its 'bystander' activities (10, 31).
  • CaP is ideally suited for the first test of efficacy of this idea because this non-essential organ expresses a wide array of unique antigens and highly accessible to gene transfer by using digital or transrectal ultrasound guidance (45).
  • primary CaP is relatively slow growing and thus sequential gene therapy approaches can be incorporated safely into treatment strategies.
  • Serum prostate-specific antigen (PSA) can be easily used to monitor treatment response.
  • PSA prostate-specific antigen
  • the strategy may also prove effective against CaP cells that are androgen-independent, since mda-7/IL-24 causes release of tumor antigens from CaP regardless of their androgen sensitivity.
  • this approach may be useful in combination with androgen-deprivation therapy and can be tested in hormone- refractory CaP.
  • Fisher PB Gopalkrishnan RV, Chada S, et al. m ⁇ i ⁇ -7/IL-24, a novel cancer selective apoptosis inducing cytokine gene: from the laboratory into the clinic. Cancer Biol Ther 2003;2:S23-37. 6. Lebedeva IV, Su ZZ, Chang Y, Kitada S, Reed JC, Fisher PB. The cancer growth suppressing gene mda-1 induces apoptosis selectively in human melanoma cells. Oncogene 2002;21 :708-18.
  • BiP/GRP78 is an intracellular target for MDA- 7/IL-24 induction of cancer-specific apoptosis. Cancer Res 2006;66:8182-91.
  • FISP IL-4-induced secreted protein
  • This Example describes systemic delivery of the virus in therapy resistant BCI-X L overexpressing tumor xenografts in nude mice using UTMD.
  • Systemic delivery of the antitumor gene mda- 7/ ⁇ L-24 by microbubbles was studied.
  • DU-145-BCI-X L DU- 145 ectopically express BcI- X L
  • Ad.m ⁇ i ⁇ -7-resistant variant of DU- 145 human prostate carcinoma cells DU-BCI-X L tumor xenografts were established on both flanks of nude mice by injecting 2 X 10 6 cells in each side of the animal.
  • Treatment was initiated when the tumor reached a size of 250 - 350 mm 3 .
  • No treatment was performed on the tumor xenografted on the left flank.
  • Animals receiving the Ad-GFP-microbubble complexes plus US treatment showed no statistically significant effect on the growth of DU-145-BCI-X L tumors.
  • Microbubbles (Ad.PEG-ElA-m ⁇ i ⁇ -7) elicited a sustained growth inhibition of the therapy resistant DU-BcI- X L tumor xenografts in both primary and distant tumors.
  • B- mode ultrasound-scan of DU-BCI-X L tumors showed dramatic volume reductions in the tumors after 2 and 4 wks of treatments with microbubble complexes and US leading to the eradication of the tumor xenograft (data not shown). Additionally, no tumor regrowth in the primary or distant sites was evident in microbubble complex and US-treated DU-BCI-X L animals after an additional three weeks post-treatment.
  • Example 4 A major challenge for effective gene therapy using Ads or recombinant proteins is the ability to specifically deliver the therapeutic directly into diseased tissue without exposure to the immune system, particularly with a systemic approach in an immune competent animal model.
  • Progress in gene therapy has been hampered by concerns over the safety and practicality of viral vectors, particularly for intravenous delivery, and the inefficiency of currently available non- viral transfection techniques, we have developed a novel targeted- delivery approach that includes ultrasound (US) contrast agents (microbubbles) to deliver effective therapeutic reagents Ad.mda-7 or GST-MD A-7 (melanoma differentiation associated gene-7/interleukin-24), followed by ultrasound-targeted microbubble destruction (UTMD) to develop a prostate-specific therapy in a prostate cancer immune competent transgenic mouse model, Hi-Myc.
  • US ultrasound
  • UTMD ultrasound-targeted microbubble destruction
  • Protocol for systemic delivery of Ad.mda-7 into prostate of immunocompetent prostate cancer mouse model (Hi-Myc): [0158] Preparation of microbubbles (MBs), US platform and UTMD. Targeson (Targeson) custom synthesis US contrast agent (perfluorocarbon MBs, encapsulated by a lipid monolayer and poly(ethyleneglycol) stabilizer) were prepared following manufacturer's instructions. MBs were reconstituted in the presence or absence of ImI of 0.5 x 10 12 plaque-forming units of Ad.vec or Ad.mda-7 and complement was achieved by incubating MBs/ Ad complex with Human complement purchased from Sigma.
  • mice received injections of 100 ⁇ l of MBs with Ads through the tail vein twice a week for 4 weeks.
  • the mice were divided into three groups I) Ad.5/3-vec, II) Ad.5/3-mda-7, III) BI-97C1 (a novel McI-I inhibitor), (IV) Ad-5/3-mda-7+ BI-97C1 group.
  • US was performed with a SonoSite scanner (SonoSite) equipped with the transducer L25 set at 0.7 Mechanical Index, 1.8 MPa for 10 minutes in the ventral side of mice in the prostatic area.
  • the Hi-Myc mice were sacrificed and the prostate was dissected and weighed.
  • the harvested prostate were preserved in neutral buffered formalin at 4 0 C before embedding in paraffin for immunohistochemical analysis.
  • Our data supports efficient, targeted transgene delivery of mda-7/IL-24 in the prostate of Hi- Myc mice using UTMD (Fig. 18).
  • mice were sacrificed 4 weeks after treatment and tumor progression was observed followed by dissection of animals (Fig. 19).
  • the GST, GST/microbubble with US, GST-MD A-7 and GST-MD A-7/microbubble without US groups did not induce any tumor regression (Fig. 19A, 19B, 19C, 19D), whereas the GST-MD A-7/microbubble with US group showed profound tumor regression (Fig. 19E).

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Abstract

L'invention porte sur l'administration de virus aidée par microbulle. En particulier, l'invention porte sur des procédés pour cibler un virus dans des cellules cancéreuses dans un animal immunocompétent, par l'administration d'un virus à réplication sélective à l'animal immunocompétent et la rupture des microbulles dans un emplacement de l'animal comprenant des cellules cancéreuses. Le virus est englobé dans une suspension de microbulles et la surface de la suspension ne comprend aucun virus. L'invention porte également sur une suspension de microbulles comprenant un virus à réplication sélective qui est englobé dans une suspension de microbulles, qui ne comprend aucun virus sur la surface de la suspension.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014093270A1 (fr) * 2012-12-10 2014-06-19 Virginia Commonwealth University Virus terminateur de cancer à tropisme modifié (ad.5/3 ctv ; ad.5/3-ctv-m7)
US20160106866A1 (en) * 2013-06-04 2016-04-21 The Johns Hopkins University Tripartite cancer theranostic nucleic acid constructs
US9974816B2 (en) 2012-11-02 2018-05-22 N.V. Nutricia Synbiotics combination for brain improvement
CN110585127A (zh) * 2019-07-15 2019-12-20 三峡大学 靶向pd-l1微泡的制备方法及在制备抑制宫颈癌的药物上的应用

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US20040151702A1 (en) * 2001-04-20 2004-08-05 Rainer Marksteiner Production and use of a suspension composition comprising an ultrasound contrast medium
US20080063604A1 (en) * 2006-09-12 2008-03-13 Temple University - Of The Commonwealth System Of Higher Education Method and composition for the site-selective gene delivery using viruses
US20080145937A1 (en) * 2006-09-22 2008-06-19 Baylor Research Institute In Vivo Transformation of Pancreatic Acinar Cells into Insulin-Producing Cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040151702A1 (en) * 2001-04-20 2004-08-05 Rainer Marksteiner Production and use of a suspension composition comprising an ultrasound contrast medium
US20080063604A1 (en) * 2006-09-12 2008-03-13 Temple University - Of The Commonwealth System Of Higher Education Method and composition for the site-selective gene delivery using viruses
US20080145937A1 (en) * 2006-09-22 2008-06-19 Baylor Research Institute In Vivo Transformation of Pancreatic Acinar Cells into Insulin-Producing Cells

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9974816B2 (en) 2012-11-02 2018-05-22 N.V. Nutricia Synbiotics combination for brain improvement
WO2014093270A1 (fr) * 2012-12-10 2014-06-19 Virginia Commonwealth University Virus terminateur de cancer à tropisme modifié (ad.5/3 ctv ; ad.5/3-ctv-m7)
US10314870B2 (en) 2012-12-10 2019-06-11 Virginia Commonwealth University Tropism modified cancer terminator virus (Ad.5/3 CTV;AD.5/3-CTV-M7)
US20160106866A1 (en) * 2013-06-04 2016-04-21 The Johns Hopkins University Tripartite cancer theranostic nucleic acid constructs
US10166300B2 (en) * 2013-06-04 2019-01-01 Virginia Commonwealth University Tripartite cancer theranostic nucleic acid constructs
CN110585127A (zh) * 2019-07-15 2019-12-20 三峡大学 靶向pd-l1微泡的制备方法及在制备抑制宫颈癌的药物上的应用

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